Blood platelets are required for hemostasis and mediate diverse pathologic processes, including thrombosis and inflammation. Although the origin of platelets in megakaryocytes (MKs) was recognized over a century ago, cellular and molecular mechanisms of platelet assembly and release remain largely obscure. Current understanding and experimental approaches build on a combination of cell biological studies, genetic analysis in mice, and appreciation of congenital human thrombocytopenias such as the May-Hegglin anomaly and other Myh9-related disorders. At the conclusion of an elaborate maturation process, MKs undergo dramatic morphogenesis to extend long, branched structures called proplatelets. Blood platelets are assembled de novo within these cytoplasmic extensions and ultimately released into the circulation. As a result of NHLBI funding through an R01 award that is now in its 9th year, our group has played a prominent part in investigating the mechanisms of this remarkable process, which depends on activity of the transcription factor NF-E2. We seek to renew funding to continue with studies that will significantly advance understanding of how MKs produce and release platelets. First, we recently reported on the surprising result that the Myh9 gene product, non-muscle myosin heavy chain IIA, regulates proplatelet formation negatively and seems to receive signals through the small-GTPase Rho. Our preliminary studies suggest 2 specific hypotheses: (1) that myosin-IIA deficiency promotes precocious platelet assembly within immature MKs, and (2) that myosin-IIA inhibition of platelet release is normally lifted when mature MKs encounter the chemokine Sdf-1/CXCL12, which down-regulates MK Rho activity. In Specific Aim 1 we propose to test these hypotheses critically, using multi-photon intravital microscopy and biochemical and functional analysis of signal transduction in cultured primary mouse MKs. Second, although NF-E2 is a seminal transcriptional regulator of late MK maturation and platelet biogenesis, a satisfactory understanding of its mechanisms and transcriptional targets has been elusive. We have combined chromatin immunoprecipitation with hybridization to tiled genome arrays (ChIP-chip methodology) to begin to identify the nearly complete complement of genes that NF-E2 may regulate in MKs. In Specific Aim 2 we propose to solidify and extend our preliminary ChIP-chip findings to characterize the NF-E2 [unreadable]cistrome[unreadable] and the breadth of cellular processes recruited in thrombopoiesis. Our studies have already started to generate specific and powerful hypotheses about particular NF-E2- binding cis-elements in MK-expressed genes. In Specific Aim 3 we will use cell-based reporter assays and locus-targeted mice to confirm the requirements for particular NF-E2 cis-elements in MK gene regulation. The sum of these studies should lead to new insights into physiologic and molecular control of thrombopoiesis.